The present invention is related to the biological control of plant diseases caused by Fusarium species. Specially, it relates to biocontrol compositions comprising a mixture of at least one microorganism which is an antagonist against plant pathogens and an appropriate carrier, as well as to a process for control of the plant pathogen and to increase yield. The invention includes as a plant pathogen the pathogenic fungus Gibberella zeae (anamorph Fusarium graminearum), and as antagonist microorganisms the novel isolates of Pantoea agglomerans (Embr. 1494, Accession ATCC PTA 3460) and of Bacillus megaterium (Embr. 9790, Accession ATCC PTA 3461).
Fruit, vegetables, and plants are all susceptible to attack by fungi, resulting in loss of crops, decreased shelf-life of produce, and ultimately higher costs for consumers. Many fungi are known pathogens in several diseases which harm or destroy crops worldwide. Examples of such fungi include those belonging to the genera Rhizoctonia, Pythium, Gaeumannomyces, and Fusarium.
For a number of years, it has been known that various microorganisms exhibit biological activity useful in controlling plant diseases. Although progress has been made in the field of identifying and developing biological pesticides for controlling plant diseases of agronomic and horticultural importance, most of the pesticides in use are still synthetic compounds. Many of the chemical fungicides are carcinogenic agents and, therefore, toxic to wildlife and other non-target species. In addition, pathogens may develop resistance to chemical pesticides. In fact, the fungicides, considered the major weapon in combating plant diseases, are often ineffective and pose hazards to humans and the environment. Biological control offers an attractive approach as compared with synthetic chemical fungicides. Biopesticides (living organisms and the compounds which are naturally produced by these organisms) can be safer, more biodegradable, and less expensive to develop. In addition, they are highly desired for integrated pest management programs in agriculture, public health, and urban settings.
The agricultural use of Bacillus megaterium has been reported for disease control in rice and cotton inhibition as seed treatment but not as foliar sprays. U.S. Pat. No. 5,403,583 discloses a Bacillus megaterium, ATCC 55000, and a method to control the fungal plant pathogen Rhizoctonia solani as seed treatment. Islam and Nandi also disclosed a Bacillus megaterium with antagonism to Drechslera oryzae, the causal agent of rice brown spot (Journal of Plant Diseases and Protection. 92(3): 241-246 (1985) and a Bacillus megaterium with in vitro antagonism against Drechslera oryzae, Alternaria alternata and Fusariun roseum (Journal of Plant Diseases and Protection. 92(3): 233-240 (1985). They mentioned three components in the culture filtrate. The most active antibiotic was highly soluble in water and methanol with a UV peak at 255 nm and a shoulder at 260 nm, which proved to be a polyoxin-like lipopeptide. And, Cook (Proceedings Beltwide Cotton Production-Mechanization Research Conference, Cotton Council, Memphis, p. 43-45 (1987) disclosed the use of a suspension of Bacillus megaterium to reduce the number of plants killed by Phymatotrichum omnivorum, a cause of cotton root rot. Antibiotic production of B. megaterium has also been recorded by Berdy (CRC Handbook of Antibiotic Compounds, Vols. I-XIV. CRC Press, Inc. Boca Raton. Fla. 1980-87), who reported the production of low-mammalian toxic peptide antibiotics such as ansamitocin-PDM-O, bacimethrin, megacin, pentapeptide, and homopeptides.
U.S. Pat. No. 5,494,819 describes a pure culture of Pantoea agglomerans having all of the characteristics of FERM BP-3511 which is identified by growth, morphology, physiology, utilization of carbon sources and various specific enzymatic tests involving enzymes as lysine decarboxylase, arginine dihydroxylase, phenylalanine deaminase and ornithine decarboxylase. In addition, the disclosed pure culture of Pantoea agglomerans is characterized by the required production of lipopolysaccharides to which the inventors attribute an immunity-stimulating activity. In other words, according to this document, Pantoea agglomerans is used to obtain substances to be used in pharmaceuticals.
U.S. Pat. No. 5,766,926 discloses a method comprising the steps of applying to the pulpwood or pulp substrate a bacterial inoculum of at least one of the species selected from the group consisting of Pseudomonas fluorescens, Pantoea (Enterobacter) agglomerans, Bacillus cereus, and Xanthomonas campestris and maintaining the substrate under conditions which allow bacterial growth for a time sufficient to effect a reduction in the resin component of the substrate by the bacteria. It is mentioned that the source of the Pantoea (Enterobacter) agglomerans isolate used in the method, identified by the NRRL Accession No.B21509, is Brazil.
It is known that the genus Fusarium contains species which may cause diseases of wither and blight that occur during the growth of plants and damages not only the host but also other kinds of plants. It is supposed that fusaric acid is the principal agent that brings about these diseases. Fusaric acid (5-n-butylpicolinic acid) is known to be a non-specific toxin which is produced by the metabolism of almost all plant pathogenic Fusarium fungi (Wood, R. K. et al. 1972. xe2x80x9cPhytotoxins in plant diseasesxe2x80x9d. Academic Press. New York; Durbin, R. D. 1982. xe2x80x9cToxins in plant diseasesxe2x80x9d. Academic Press. New York). In the document EP 257 756, referring to the prevention of Fusarium diseases and microorganisms therefor, the inventors proposed to prevent such diseases by using microorganisms belonging to the genera Cladosporium and Pseudomonas which decompose fusaric acid. EP 441 520 relates also to detoxifying fusaric acid microorganisms, and Klebsiella oxytoca HY-1 (FERM BP-3221) is particularly mentioned.
In the document WO 92018613, it is suggested to control plant diseases caused by fungi of the genera Rhizoctonia, Pythium, and Fusarium by using a new strain of Pseudomonas fluorenscens, a seed or soil treatment but not foliar sprays.
WO 9905257, referring to biocontrol for plants with Paenibacillus macerans, Pseudomonas putida, and Sporobolomyces roseus, describes the use of isolates of these microorganisms to impart pathogen protection to plants, particularly against plant diseases caused by fungi, such as Fusarium oxysporum, Fusarium graminearum, Fusarium monilforme, Cochliobolus sativus, Collectotrichum graminicola, Stagonospora nodorum, Stagonospora avenae, Stenocarppela maydis, and Pyrenophora tritici-repentis. In this case, pathogen protection was achieved by either seed treatment or foliar sprays.
Fusarium graminearum Schw. (Teleomorph=Gibberella zeae Schw. Petch.) is the Fusarium species most frequently responsible for scab of wheat and barley in Brazil. This disease, also known as Fusarium Head Blight (FHB), is responsible for major losses which vary from 10% (see Luz, W. C. da. 1984. xe2x80x9cYield losses caused by fungal foliar wheat pathogens in Brazilxe2x80x9d. Phytopathology. 74:1403-1407); to 54% (Picinini, E. C. and Fernandes, J. M. C. 1994. xe2x80x9cControle quimico da Gibberella zeae em trigo pelo uso de fungicidas inibidores da sintese do ergoterolxe2x80x9d. Fitopatol. Brasileira 19 (Supl.):273). At present, available and affordable control measures, such as resistant varieties, cultural practices, and foliar fungicides, are only partially effective.
Only modest levels of resistance have been deployed in cultivars in commercial fields; the most widely grown cultivars are often most susceptible. Furthermore, the benefit of crop rotation as a control measure is reduced by the wide host range of the pathogen, especially on grasses (Costa Neto, J. P. da. 1976. xe2x80x9cLista de fungos sobre gramineas (capins e cereais) no Rio Grande do Sulxe2x80x9d. Revista da Faculdade de Agronomia. UFRGS. 1:43-78; Luz, W. C. da. 1982. xe2x80x9cDiagnose das Doencas da Cevadaxe2x80x9d. Passo Fundoxe2x80x94EMBRAPA-CNPT, 24p. (Circular Txc3xa9cnica nxc2x0. 2)). Treatment with foliar fungicides remains the most important (Picinini and Fernandes, 1994) and recommended (Reunixc3xa3o da Comissxc3xa3o Sul-Brasileira de Pesquisa de Trigo, 2000) tool for reducing scab in Brazil, despite its shortcomings as a control measure. The use of certain effective fungicides has been restricted in some countries because application at late developmental stages, that is, during heading and flowering, can result in chemical residues in the harvested grain. Biological control is an additional strategy that may eventually play an important role in an integrative approach to scab management of cereals.
Screening of microorganisms to control wheat scab was initiated in Brazil in the 1980""s (Luz, W. C. da. 1988. xe2x80x9cBiocontrol of fungal pathogens of wheat with bacteria and yeastsxe2x80x9d. Page 348 in: 5th International Congesss of Plant Pathology, Kyoto, Japan. (Abstr.)). At the beginning, over 300 bacteria and yeasts isolated from wheat were screened in vitro against F. graminearum. This work was followed by that of Perondi et al. (Perondi, N. L., Thomas, R. and Luz, W. C. da. 1990. xe2x80x9cAntagonistas potenciais de Fusarium graminearumxe2x80x9d. In: Anais do 2xc2x0 Simpxc3x3sio de Controle Bixc3x3lgico, Brasilia, D. F., p. 128.(Abstr.); Perondi, N. L., Thomas, R. and Luz, W. C. da. 1990. xe2x80x9cControle microbiano da giberela do trigo em campoxe2x80x9d. In: Anais do II Simpxc3x3sio de Controle Biolxc3x3gico, Brasilia, D F. P.129(Abstr.); Perondi, N. L., Luz, W. C. da. and Thomas, R. 1996. xe2x80x9cControle microbiolxc3x3gico da giberela do trigoxe2x80x9d. Fitopatol. Brasileira 21:243-249) in which microbial strains were tested for their antagonistic action against the pathogen. Potential antagonists were selected by the funnel method (Luz, W. C. da. 1990. xe2x80x9cMicrobiological control of Bipolaris sorokiniana xe2x80x98in vitroxe2x80x99xe2x80x9d. Fitopatol. Brasileira 15:246-247) which compared the effect of individual test organisms on the radial growth of F. graminearum. Promising isolates were further tested in the greenhouse and in the field for their ability to control wheat scab. Individual bioprotectants significantly diminished the severity of the disease under field conditions, raising the yield of wheat between 7 and 31% when compared to non-treated plants.
Besides the selection of the bioprotectants, it is important to overcome several difficulties related to constraints on their application to the ears of wheat and barley at flowering such as the timing of application, inoculation technology, physiological state of the organisms, spike colonization, survival of the organisms under the harsh environmental conditions, variability of biocontrol from year to year, fermentation, formulation, and storage. The partial control of any tactics to protect against FHB up to this moment indicates that the integration of protection measures would provide the best disease management.
From 1988 up to now, thousands of microorganisms have been tested for scab control. Some workers have been investigating antagonists to control FHB (Khan, N. J., Schisler, D. A., Boehm, M. J., Lipps, P. E., Slininger, P. J. and Bothast, R. J. 1998. xe2x80x9cBiological control of scab of wheat incited by Giberella zeaexe2x80x9d. Pages 45-46 in: Proceedings of the 1998 National Fusarium Head Blight Forum, Michigan State University, University Printing, East Lansing. Mich.; Khan, N. J., Schisler, D. A., and Boehm, M. J. 1999. USDA-ARS, Ohio State University cooperative research on biologically controlling Fusarium Head Blight: 2. Influence of pathogen strain, inoculum spray sequence and inoculum spray time. Pages 56-59 in: Proceedings of the 1999 National Fusarium Head Blight Forum, Michigan State University, University Printing, East Lansing, Mich.; Boehm, m. J., Khan, N. J., and Schisler, D. A. 1999. USDA-ARS, Ohio State University cooperative research on biologically controlling Fusarium Head Blight: 3. Field testing of antagonists. Pages 45-48 in: Preceedings of the 1999 National Fusarium Head Blight Forum, Michigan State University, University Printing, East Lansing, Mich.; Luo, Y. and Bleakley, B. 1999. xe2x80x9cBiological control of Fusarium Head Blight (FHB) of wheat by Bacillus strainsxe2x80x9d. Pages 78-81 in: Proceedings of the 1999 National Fusarium Head Blight Forum, Michigan State University, University Printing, East Lansing, Mich.; Schisler, D. A., Khan, N. J., and Boehm, M. J. 1999. USDA_ARS, Ohio State University cooperative research on biologically controlling Fusarium Head Blight: 1. Antagosist selection and testing on durum wheat. Pages 78-81 in: Proceedings of the 1999 National Fusarium Head Blight Forum, Michigan State University, University Printing, East Lansing, Mich.; Stockwell, C. A., Luz, W. C. da., and Bergstrom, G. C. 1997. xe2x80x9cBiocontrol of wheat scab with microbial antagonistsxe2x80x9d. Phytopathology 87:S94.(Abstr.); Stockwell, C. A., Bergstrom, G. C., and Luz, W. C. da. 1999. xe2x80x9cSelection of microbial antagonists for biological control of Fusarium Head blight of wheatxe2x80x9d. Pages 82-84. in: Proceedings of the 1999 National Fusarium Head Blight Forum, Michigan State University, University Printing, East Lansing, Mich.; Stockwell, C. A., Bergstrom, G. C., and Luz, W. C. da.2000. xe2x80x9cIdentification of bioprotectants for biological control of Gibberella zeaexe2x80x9d in:. Proceedings of FHB Forum), under greenhouse or field conditions. Some strains have reduced the FHB severity and significantly reduced vomitoxin contamination in grains (Stockwell et al., 1997, 2000). Table 1 illustrates the chronology of researches on the biocontrol of FHB.
According to the present invention, microbiological agents are provided for control of certain diseases of wheat and other cereals caused by Fusarium species, including Fusarium Head Blight (FHB) of wheat and other cereals. Moreover, these agents can also improve yield of said wheat plants and cereals. Specifically, these agents are novel isolates of Pantoea agglomerans and of Bacillus megaterium that exhibit the property of inhibiting fungal pathogens, particularly those produced by Fusarium species.
The first embodiment of the invention refers to a biocontrol composition comprising a mixture of at least one microorganism which is antagonist against plant pathogens and a carrier for said at least one microorganism, wherein said at least one microorganism is a bacteria selected from the group consisting of Pantoea agglomerans and Bacillus megaterium and said at least one microorganism is present in an amount effective for inhibiting plant pathogen development.
A second embodiment is related to a process for controlling the plant pathogen development on wheat and cereal plants by applying a composition containing a carrier and at least one microorganism which is an antagonist against plant pathogens selected from the group of bacteria consisting of Pantoea agglomerans and Bacillus megaterium in an amount effective to inhibit plant pathogen development on said plant.
The third embodiment is related with a process for increasing plant yield characterized by a step of applying, particularly by spraying, to the plant a composition containing a carrier and at least one microorganism selected from the group of bacteria consisting of Pantoea agglomerans and Bacillus megaterium in an amount effective to increase yield of said plants or plants resulting from treated seeds.
For purposes of clarity and a complete understanding of the invention, the following terms are defined.
xe2x80x9cPlantsxe2x80x9d is used to mean the head part of the plant to be treated.
xe2x80x9cPantoea agglomerans (Embr. 1494)xe2x80x9d means the bacterium isolate which was isolated by Embrapa and identified by the code xe2x80x9cEmbr. 1494xe2x80x9d.
xe2x80x9cBacillus megaterium (Embr. 9790)xe2x80x9d means the bacterium isolate which was isolated by Embrapa and identified by the code xe2x80x9cEmbr. 9790xe2x80x9d.
xe2x80x9cCFUxe2x80x9d refers to the abbreviation of Colony Forming Unity which is frequently used to express the concentration of microorganisms present in a composition.
Microorganisms usable in the present invention were identified by the following procedure: (i) screening plants or agricultural commodities (e.g. the surface(s) of said plant or agricultural commodity) for the presence of useful microorganisms; (ii) recovering (e.g. by washing or rising from the plant or agricultural commodity) and isolating said microorganism(s); and (iii) testing said microorganism(s) for antagonistic activity against plant pathogens. However, it should be understood that said microorganism(s) may be obtained from sources other than said plants or agricultural commodities.
The isolates of the present invention, Accession No. ATCC PTA 3460 (Embr. 1494) deposited at the ATCC (Manassas, Va.) on May 29, 2001, and Accession No. ATCC PTA 3461 (Embr. 9790) deposited at the ATCC (Manassas, Va.) on May 29, 2001, were obtained from wheat or corn plant parts, such as healthy leaves, seeds or roots by repeatedly washing the plant parts with water. The organisms were thereafter plated and grown on any nutritionally rich medium sufficient to support growth of the organisms. Preferably, the medium is nutrient agar. ATCC PTA 3460 was identified as a novel isolate of Pantoea agglomerans (Embr. 1494) and ATCC PTA 3461 was identified as a novel isolate of Bacillus megaterium (Embr. 9790).
Isolate ATCC PTA 3461 (Embr. 9790) of B. megaterium has the following characteristics: it is a Gram-positive rod, spore-forming bacteria, and the bacterial identification was accomplished based on 16S rRNA gene sequence similarity (made by Microbe Inotech Laboratories, Inc on Apr. 21, 2000 by using PE Applied Biosystem""s MicroSeq(trademark) microbial identification software and database) demonstrating that the isolate is novel and belongs to the species Bacillus megaterium (details about this characterization method may be found in Stackebrandt, E. and Goebel, B. M. 1994. xe2x80x9cTaxonomic Note: A Place for DNAxe2x80x94DNA Reassociationxe2x80x9d; and 16S rRNA Sequence Analysis in the Present Species Definition in Bacteriology. Int. J. Syst. Bacteriol. 44:846-849).
Isolate ATCC PTA 3460 (Embr. 1494) of P. agglomerans has the following characteristics: it is a Gram-negative bacteria; and, based on fat acid analysis (CG FAME method), has a similarity coefficient of 0.648 and distance coefficient of 3.310 (a good match is one with a similarity coefficient greater than 0.5 and a distance coefficient of less than 7) made by the same laboratory.
Growth of isolates ATCC PTA 3460 (Embr. 1494) and ATCC PTA 3461 (Embr. 9790) may be effected under aerobic condition at any temperature satisfactory for growth of the microorganisms, i.e., from about 10xc2x0 C. to about 30xc2x0 C. The preferred temperature range is 20xc2x0 C. to 25xc2x0 C. The pH of the nutrient medium is about neutral, i.e., 6.6 to 7.3. The incubation time is that time necessary for the isolates to reach a stationary phase of growth, preferably between 40 and 60 hours. Growth of isolates ATCC PTA 3461 (Embr. 9790) (B. megaterium) and ATCC PTA 3460 (P. agglomerans) (Embr. 1494) is preferably achieved at a temperature range of 21xc2x0 C. to 23xc2x0 C., with an incubation time of 45 to 50 hours, such that the cells are in the logarithmic phase of growth.
Isolates ATCC PTA 3461 (Embr. 9790) (B. megaterium) and ATCC PTA 3460 (Embr. 1494) (P. agglomerans) may be grown in any conventional test tube or shake flask for small fermentation runs. For large scale operations, the culture may be carried out in a suitable fermentation tank, under appropriate conditions provided by agitating and aerating the inoculated liquid medium. Following incubation, the isolates are harvested by conventional sedimentary methods (e.g. centrifugation) or filtering. Cultures are stored on nutrient agar at 4xc2x0 C., but also at much lower temperature such as xe2x88x92170xc2x0 C.
The bacteria of the present invention are useful to control plant pathogens by using, for example, air spraying.
The microorganisms of the present invention may be applied to wheat plants or other cereals in combination with various liquid and/or solid carriers and additives, including combination with fungicides.
In the liquid form, e.g. solutions or suspensions, the microorganisms may be mixed or suspended in water or in aqueous solutions. Suitable liquid diluents or carriers include water, aqueous solutions, petroleum distillates, or other liquid carriers.
Solid compositions can be prepared by dispersing the antagonist microorganisms in and on an appropriately divided solid carrier, such as peat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceous earth, fuller""s earth, pasteurized soil, and the like. When such formulations are used as wettable powders, biologically compatible dispersing agents such as non-ionic, anionic, amphoteric, or cationic dispersing and emulsifying agents can be used.
In a preferred embodiment, the compositions contemplated herein prevent attack by Fusarium diseases upon plants, particularly cereal plants, such as wheat, barley, and corn and, when used in sufficient amounts, to act as fungi antagonist. They have a high margin of safety because they do not burn or injury the plant.
The compositions of the invention are so chemically inert that they are compatible with substantially any other constituents of the spray schedule. They may also be used in combination with biologically compatible pesticidal active agents as for example, herbicides, nematocides, fungicides, insecticides, and the like. They can also be used in combination with plant growth affecting substances, such as fertilizers, plant growth regulators, and the like, provided that such compounds or substances are biologically compatible.
The active constituents are used in a concentration sufficient to inhibit plant pathogen development of the targeted plant pathogen when applied to the cereal plant. As will be apparent to a skilled in the art, effective concentrations may vary depending upon such factors as: (a) the type of the plant or agricultural commodity; (b) the physiological condition of the plant or agricultural commodity; (c) the concentration of pathogens affecting the plant or agricultural commodity; (d) the type of disease injury on the plant or agricultural commodity; (e) weather conditions (e.g. temperature, humidity); and (f) the stage of plant disease. According to the invention, typical concentrations are those higher than 1xc3x97102 CFU/ml of carrier. Preferred concentrations range from about 1xc3x97104 to about 1xc3x97109 CFU/ml, such as the concentrations ranging from 1xc3x9710106 to 1xc3x97108 CFU/ml. More preferred concentrations are those of from about 37.5 to about 150 mg/g of dry bacterial mass per of carrier (dry formulation) or per ml of carrier (liquid composition).
The compositions of the invention may be applied to the wheat plant or other cereals using conventional methods such as dusting, injecting, rubbing, rolling, dipping, spraying, or brushing, or any other appropriate technique which does not injury the wheat plant or other cereals to be treated. Particularly preferred is the spray method.
The following specific examples are presented to more particularly illustrate the invention and should not be construed as a limitation thereon.